Fig. 3

CDS m⁶A methylation resolves mRNA secondary structures. a The predicted minimum folding free energy (MFE) is plotted along mRNA regions surrounding the CDS RRAC motif with (pink line) or without (blue line) m⁶A modification. A sliding window with 30 nt in length and a step of 3 nt was used to calculate MFE. For each window, the central position is used for alignment. A minus position value indicates upstream of m⁶A sites, whereas a positive value indicates downstream of m⁶A sites. The m⁶A site in RRAC motif is highlighted by red. Notably, a lower MFE value indicates a higher potential for RNA secondary structures. b The GC content is plotted along mRNA regions surrounding the CDS RRAC motif with (pink line) or without (blue line) m⁶A modification. c The in vivo icSHAPE signal is plotted along mRNA regions surrounding the CDS RRAC motif with (pink) or without (blue) m⁶A modification. Notably, a higher in vivo icSHAPE signal indicates a less structured region. The right boxplot shows the average of icSHAPE signals across mRNA regions from –500 nt to 500 nt relative to the RRAC motif with (pink) or without (blue) m⁶A modification (Wilcox test, ***P < 0.001). The median of icSHAPE signals in each group is indicated by a center line, the box shows the upper and lower quantiles, whiskers shows the 1.5× interquartile range. The outliers are not shown. d The left panel shows the schematic of a dual luciferase reporter with a sandwiched secondary structure derived from MALAT1 (2556–2587). Both UU → CC and A → G mutants are also shown. The m⁶A site is highlighted by red. The right panel shows the ratio of Rluc/Fluc in transfected cells expressing wild type or indicated mutants. Error bars, mean ± s.e.m.; Single-tailed t test, n = 4, *P < 0.05, **P < 0.01. e The ratio of Rluc/Fluc in transfected cells expressing wild type or mutant reporters, with either METTL3 or METTL14 knockdown. Error bars, mean ± s.e.m.; single-tailed t test, n = 4, *P < 0.05, **P < 0.01. Source data are provided as a Source Data file.